Understanding factors that mediate the transition of hepatocellular degeneration to necrosis (i.e., reversible to irreversible injury) is central to our understanding of the maintenance of liver cell integrity. In light of studies that have suggested that cyclooxygenase metabolites of arachidonic acid were protective against hepatocyte injury, whereas lipoxygenase metabolites accelerated necrosis, we hypothesize the following: Progressive hepatocellular injury results in the accumulation of free arachidonate available for metabolism and the transition from reversible to irreversible injury is marked by an augmented production of metabolites that promotes cellular necrosis. To examine this hypothesis, a model of anoxic injury was established in isolated hepatocytes. A reversible phase of injury was distinguished morphologically and biochemically from subsequent irreversible injury. Exogenous arachidonic acid administration augments the degree of injury induced by 3 hours of anoxia, an effect preventable by pretreatment with the metabolic inhibitor BW755C, an inhibitor of both cyclooxygenase and lipoxygenase pathways of arachidonate metabolism. Further, indomethacin, a selective cyclooxygenase inhibitor, or BW755C given alone diminished the degree of anoxic injury in the absence of erogenous arachidonate administration. This suggests that endogenous metabolites may have a physiologically significant role in the progression of hepatocyte injury. Analysis of the fate of 3H- arachidonic acid in isolated hepatocytes, by lipid extraction and separation via TLC and HPLC, revealed that anoxic injury resulted in a decreased rate of esterification and an augmented production of oxidation products, many of which were covalently bound into membrane lipids. The proposed studies will examine in detail the alteration of arachidonic acid metabolism by anoxic injury and the relationship of these alterations to hepatocellular homeostasis. The influence of arachidonic acid metabolites derived from nonparenchymal cells on hepatocyte integrity will also be examined. In addition, the role of arachidonic acid metabolism in hepatocellular regeneration will be studied. Finally, studies will be performed using the Hep G2 cell line, with the goal of isolating mutant cell lines either more tolerant or more resistant to anoxic injury. Such mutant cell lines would be powerful tools to examine the mechanisms involved in the transition from reversible to irreversible injury.